Am4611

EGFP-IP₃R1 HeLa cells were plated in clear-bottomed 96-well plates (Greiner Bio-One) coated with fibronectin (10 µg ml⁻¹). Cells were transfected with siRNA directed against GFP (50 nM, #AM4626, ThermoFisher) or a nonsilencing control siRNA (50 nM, #AM4611, ThermoFisher) using siPORT NeoFX transfection reagent (ThermoFisher, 220 ng siRNA per µl reagent). After 72 h, cells were washed with HEPES-buffered saline (HBS: 135 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 1.5 mM CaCl₂, 11.6 mM HEPES, 11.5 mM glucose; pH 7.3), loaded with Fluo-8 by incubation in HBS with Fluo-8 AM (2 µM, 60 min, 20 °C), washed and incubated in HBS (60 min, 20 °C) before experiments. A FlexStation 3 microplate reader (Molecular Devices) was used to measure Fluo-8 fluorescence at 20 °C. After addition of BAPTA in Ca²⁺-free HBS (final BAPTA and CaCl₂ concentrations = 2.5 and 0.75 mM, respectively; free [Ca²⁺]<40 nM), histamine (100 µM) was added to stimulate IP₃ formation through endogenous H₁ histamine receptors. Fluorescence was collected (using SoftMax Pro, Molecular Devices) and calibrated to cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) as described^{63 (link)}.

Silencer Select siRNAs targeting mouse Lrig1 (AM16708, 4390771) and negative control (AM4611, AM4613) were purchased from Thermo Fisher Scientific. In all, 1 × 10⁶ cells of iT_reg cells per nucleofection reaction were resuspended in Mouse T cells Nucleofector Solution (100 μl per nucleofection) (Lonza) at room temperature. In all, 50, 150 nM of Lrig1 siRNA (siLrig1) or 150 nM of control siRNA (siControl) was combined with 100 μl of the cell suspension and transferred into the nucleocuvettes of the 4D-Nucleofector X unit (Lonza). The Nucleocuvettes were placed into the 4D-Nucleofector X unit and pulsed with the Nucleofector program DN-100. The nucleocuvettes were incubated for 10 min at 37 °C, 5% CO_2, and then resuspended with RPMI 1640 medium. After 24 h post nucleofection, siRNA-treated iT_reg cells were stained with relevant antibodies to confirm the expression level of markers. The cells were co-cultured with eFlour 670-stained effector T cells for the suppression assay, followed by flow cytometric analysis. To measure the cell proliferation capacity, siRNA-treated iT_reg cells were cultured with IL-2 (20 ng ml⁻¹) for 72 h. Cell proliferation of each group was quantified with the gate by Flowjo V10.

For downregulation experiments, 30nM of negative control scramble (SCR siRNA) (AM4611, Thermo fisher scientific), IL32 siRNA (mixt of s17656, s17657 and s17658 (1:1:1, total 30nM), Thermo Fisher scientific) or PLA2G2A siRNA (mix of s10589, s10591 and s224271, s17658 (1:1:1, total 20nM) Thermo Fisher Scientific) was transfected with Lipofectamine 3000, as per manufacturer’s instructions. For 2D culture, transfection was performed 24 h after seeding cells and cells were transfected for 48 h till endpoint analyses. For spheroids, transfection mix was supplemented in the media at the time of seeding to facilitate maximum uptake of siRNA. HepG2+ LX-2 spheroids were grown for 96 h till harvesting endpoint, while PHH and PHH + PHHSC spheroids were left to form for 7 days until endpoint analysis. Even with media changes, no additional transfections were performed. RNA from cells and spheroids were extracted with the RNeasy Plus Mini Kit (Qiagen) and reverse transcribed using high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Gene expression was assessed by Real-Time qPCR using master mix (Life Technologies) and TaqMan probes for IL32 and PLA2G2A according to the manufacturer’s protocol. All reactions were performed in triplicate. Data were analyzed using the 2^−ΔΔCt method normalized to beta actin.